Protein processing and development requires high efficiency processing with the minimum number of steps and the maximum output to achieve the required purity. Protein separation and purification processes present unique challenges due to the variety of proteins, the different nature of possible contaminants and/or impurities associated with each protein preparation, and the large quantity of proteins usually needed for the production of biopharmaceuticals. Conventional purification technologies generally involve a series of purification steps with the objective of isolating a single protein target. With each step, the yield decreases and manufacturing costs increase. Protein separation and purification costs typically represent over 50% of the total manufacturing costs of all therapeutic proteins.
Affinity chromatography is one of the most important separation techniques at the heart of the drug discovery and process development. The more selective the affinity step(s), the greater the efficiency of the entire enterprise, which is a critical requirement in protein fractionation experiments. Affinity chromatography finds a number of practical applications in purification, detection and removal of target molecules from multicomponent streams. Affinity chromatography is based on specific, three-dimensional interactions between target molecules and entities to which they bind (i.e., ligands). Ligands can be isolated or generated for binding in a specific and reversible manner to practically any target molecule. Potential ligands include biological molecules such as proteins, antibodies, peptides and the like, and specifically designed or selected synthetic ligands. Libraries of millions of potential ligands may be generated using combinatorial synthesis techniques, many of which are well known in the art (see, for example, Lam et al., Nature: 354, 82-84 (1991)). To aid in separation of target molecules from a sample, ligands can be affixed to a solid support matrix, such as individual particles (e.g., chromatography resin beads) or contiguous supports (e.g., arrays). Ligands immobilized on a solid support matrix can then be employed to purify targets from complex solutions.
Perhaps the greatest success of affinity chromatography at scale has been achieved in the field of biopharmaceutical monoclonal antibody purification. The demand for Protein A resin is more than 10,000 liters annually and increasing at 50% per year, representing a Protein A adsorbent market in excess of $50 Million U.S. in 2002. The use of immunoaffinity chromatography enables the production of both plasma-derived and recombinant coagulation factors VIII and IX as well as other plasma proteins and biopharmaceuticals from natural and recombinant sources.
One of the most powerful forms of modern affinity chromatography for use in downstream processing, however, relies not on ligands derived from natural sources such as antibodies, but on the use of highly stable synthetic affinity ligands. See, for example, Sproule et. al., New Strategy for the Design of Ligands for the Purification of pharmaceutical proteins by affinity chromatography; J. Chromatography B, 740, 17-33 (2000). This approach uses customized or designer ligands instead of using off-the-shelf compounds.
Among plasma proteins isolated in the art, albumin and gammaglobulin have particularly been targeted for medicinal purposes. Procedures commonly employed to isolate these proteins from plasma were based on the cold ethanol precipitation process developed by E. J. Cohn and co-workers during the 1940's. See Cohn et. al., Preparation and Properties of Serum and Plasma Proteins. IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids; J. Am. Chem. Soc., 68, 459-475 (1946). This process was originally developed to produce albumin in high yield but was not designed to isolate and purify the diverse array of proteins now produced from plasma. In particular the yields of minor plasma protein components by these techniques are invariably so low that the techniques are inevitably inefficient in terms of the overall fractionation yield. See, for example, U.S. Pat. No. 5,138,034 to Uemura et al.
The application of affinity chromatography to the purification of serum albumin is known in the art, see Harvey M J. In: Curling J M (ed) Methods of Plasma Protein Fractionation. Academic Press London. pp 189-200. The first reported separation of proteins from plasma dated about 30 years ago and concerned the depletion of human serum .albumin (HSA) by chromatography from plasma to enable identification and purification of low concentration proteins. Travis, J., and Pannell, R. Behring Inst. Mitt. 54: 30-32 (1974). This work was carried out using a Procion Blue dextran-Sepharose® conjugate and identified an initial problem with dye-affinity chromatography, namely the leakage of the dye into the eluate. The authors were interested in the isolation of alpha 1-antitrypsin from plasma and described the difficult separation of this protein from albumin at high ionic strength where any non-specific ion exchange binding is at a minimum.
The isolation of various other proteins from plasma is also reported. For example, the prior art methods described isolation and purification plasma protein Factor VIII and fibronectin fractions, see, for example, U.S. Pat. Nos. 4,822,872; 4,093,608; and 4,565,651. Methods for isolation and purification of antithrombin-III are disclosed in, for example, U.S. Pat. No. 3,842,061. Methods for isolation and purification of plasminogen are disclosed in, for example, Science: 170, 1095 (1970), U.S. Pat. Nos. 4,361,652 and 4,361,653. Methods for isolation and purification of immunoglobulins are described, for example, in U.S. Pat. Nos. 4,371,520 and 4,093,606. Methods for isolation and purification of hepataglobulin are described in, for example, U.S. Pat. Nos. 4,061,735 and 4,137,307. These methods, however, lack the specificity and selectivity required for isolation of proteins used in the production of multiple biopharmaceutical agents from the same starting material, e.g., human plasma.
Although the processes for isolating proteins from biological samples have provided some improvement in product quality, in terms of enhanced specific activity and purity, and also in yield or recovery, there still remains a need for further process improvement to obtain a protein concentrate in high yield and high purity with minimization of the reduction in the specific activity of the isolated proteins often associated with prior art processes. This is particularly true in situations where multiple protein targets are isolated simultaneously from a common source. This invention solves these and other long felt needs by providing methods utilizing affinity chromatography techniques to isolate and purify various proteins efficiently from biological materials, and particularly from plasma, by combining adsorption processes in pre-determined and defined sequences.